Development of new methods for investigating nuclear pore complex structure

Nuclear pore complexes (NPCs) are large, macromolecular protein channels that are the only known passageways across the nuclear envelope. As such, they serve highly important regulatory roles in the cell. Much of the structural information has been determined. However, correlating structure to the function is an important area of investigation. Previous atomic force microscopy (AFM) measurements have shown that a conformational change can be measured in the NPC central region on the cytoplasmic face of the nuclear envelope following specific release of calcium in the cisternal region of the nuclear envelope. Therefore, studies were initiated to measure the conformational change in the NPC on the nuclear side. AFM measurements showed a specific change in central mass position in the NPC on the nuclear face of the nuclear envelope. The studies also indicate that the change in conformation is independent of the nuclear basket. Taken with the measurements on the cytoplasmic face, the central mass appears to displace towards both faces of the nuclear envelope. This negates a simple piston-like movement of the central mass and is consistent with a mechanism of NPC release following release of cisternal calcium. Development of high-aspect ratio AFM tips was initiated to obtain additional high-resolution measurements of the central region of the NPC. Two types of high-aspect ratio tips were fabricated: FIB-carved AFM tips and single-walled nanotube (SWNT)-AFM tips. Methods were developed to perform AFM measurements on unfixed nuclear envelopes under more physiological conditions, since previous measurements were performed on fixed, dehydrated membranes, and this was successfully accomplished. The identity of the central mass is controversial, and investigating the identity requires a high resolution specific optical technique, such as near-field scanning optical microscopy (NSOM). A new method was used to fabricate fiber optic NSOM probes by chemical etching due to the higher transmission efficiencies reported for etched probes. Etched NSOM probes are comparable in resolution to the pulled probes with higher throughput. The etching method has desirable characteristics that were used to produce a new design of hybrid NSOM/AFM probe. These probes were fabricated without the use of the FIB and have higher transmission efficiencies than the previous hybrid probe design. Also, since the etched probes have much higher throughput than pulled probes, initial studies were performed towards near-field Raman spectroscopy utilizing the etched probes.